Pyrrolo-indole and pyrrolo-quinoline derivatives as prodrugs for tumour treatment

ABSTRACT

Compounds of the general formula (I) or (IA) in which X is H, Y is a leaving group, R 1  and optionally also R 3  preferably being an aromatic DNA binding subunit are prodrug analogues of duocarmycin. The compounds are expected to be hydroxylated at the carbon atom to which X is joined, by cytochrome P450, in particular by CYP1B1, expressed at high levels in tumours. The prodrug is expected to be activated preferentially in tumour cells, where it will act as a DNA alkylating agent preventing cell division.

[0001] The present invention concerns aromatic oxidation/hydroxylationactivated prodrugs, particularly anti-tumour prodrugs and those whichare specifically activated by the oxidation/hydroxylation activities ofthe cytochrome P450 family of enzymes.

[0002] Many conventional cytotoxic drugs are known that can be used fortherapeutic purposes. However, they typically suffer from the problemthat they are generally cytotoxic and therefore may affect cells otherthan those that are required to be destroyed. This can be alleviated tosome extent by the use of targeted drug delivery systems, for exampledirect injection to a site of tumourous tissue or, e.g. binding thecytotoxic agent to an antibody that specifically recognises an antigendisplayed only on the cancer cell surface. Alternatively,electromagnetic radiation may be used to cause chemical alteration in anagent at a desired site such that it becomes cytotoxic. However, all ofthese techniques have, to a greater or lesser extent, certainlimitations and disadvantages.

[0003] The compound (+)—CC-1065 and the duocarmycins are naturallyoccurring representatives of a class of DNA alkylating agents. Thenaturally occurring compounds consist of a DNA alkylating unit basedupon a pyrrolo[3,2-e]indole core, with one or two sub units, conferringDNA binding capabilities. CC-1065 and duocarmycin A comprise aspirocyclic cyclopropane group responsible for the DNA alkylationproperties. Duocarmycin B₂, C₂ and D₂ are believed to be precursors forcyclopropane actives, and comprise a substituted (by a leaving group)methyl group at the eight position on the dihydro pyrrole ring. CC-1065has been synthesised by various routes, summarised by Boger et al. inChem. Rev. 1997, 97, 787-828.

[0004] In U.S. Pat. No. 4,413,132 the first synthesis of the left handsub-unit of CC-1065 was described. The synthesis is based on a WinsteinAr-3′ alkylation in which the cyclopropane ring is introduced. In aprevious step, the A ring (of the indole core) is introduced by reactionof an aniline with an α-thiomethylester using chemistry based onGassman's Oxindole synthesis. The aniline has a protected phenolichydroxyl group ortho to the NH₂ group, which, in the final product, isbelieved to be crucial for DNA alkylation. CC-1065 has broad antitumouractivity but is too toxic against normal cells to be clinically useful.Attempts have been made to target the delivery of CC-1065 and analoguesby conjugating the drug via the DNA binding subunit to polymers, orspecific binding agents such as antibodies or biotin described in U.S.Pat. No. 5,843,937. Boger et al in Synthesis 1999 SI, 1505-1509described prodrugs of 1,2,9,9a-tetrahydrocyclopropa(c)benz[e]indol4-one,in which the cyclopropane ring-opened version of the compounds werederivatised by reaction of the phenolic group to form esters andcarbamates.

[0005] In J.Am.Chem.Soc. (1991), 113, 3980-'83 Boger et al describe astudy to identify features of CC-1065 analogues contributing to theselectivity of the DNA-alkylation. The compounds tested in vitro hadalkylating subunits based on 2,3-dihydroindole and included the6-deshydroxy analogues. These were shown to have some DNA alkylatingproperties though at concentrations 10⁴ times higher than that of the6-hydroxy compounds.

[0006] Tercel et al, in J. Org. Chem. (1999) 64, 5946-5953 describeamino analogues of CC-1065 (i.e. in which the phenolichydroxy of theB-ring is replaced by amino). These are synthesised by nitrating thebenzene ring in a late stage intermediaet having a methylol groupattached to the dihydropyrrole ring.

[0007] The present invention relates to precursors of CC-1065 and itsanalogues, which do not have the hydroxyl group in the B ring of thealkylating sub unit, and which are hence inactive as DNA alkylatingagents themselves, as well as their synthesis and intermediates usedtherein. It has been reported (Murray, G. I. et al., 15 Jul. 1997,Cancer Research, 57m 3026-3031 and WO-A-9712246) that the enzyme CYP1B1, a member of the cytochrome P450 (CYP) family of xenobioticmetabolising enzymes, is expressed at a high frequency in a range ofhuman cancers, including cancers of the breast, colon, lung, oesophagus,skin, lymph node, brain and testes, and that it is not detectable innormal tissues. This led to the conclusion that the expression ofcytochrome P450 isoforms in tumour cells provides a molecular target forthe development of new antitumour drugs that could be selectivelyactivated by the CYP enzymes in tumour cells, although no drug exampleswere given. A number of other CYP isoforms have been shown to beexpressed in various tumours. Many of the CYP's expressed in tumours arementioned in Patterson, LH et al, (1999) Anticancer Drug Des. 14(6),473486.

[0008] In WO-A-99/40056 prodrugs of styrene- and chalcone-derivativesare described. The respective hydroxylated forms of the prodrugs, formedin situ, are potent tyrosine kinase (TK) inhibitors. Inhibition of TKactivity contributes to tumour inhibition and cell destruction. Theprodrugs were shown to be activated by microsomal preparationsexpressing CYP1B1 enzyme, and to have cytotoxic activity against celllines expressing the same enzyme, whilst having much lower cytotoxicactivity against cell lines not expressing the enzyme.

[0009] The present invention is directed to a new class of prodrugswhich are expected to be hydroxylated in situ by CYP enzymes, inparticular enzymes expressed at high levels in tumours as described inPatterson LH, et al, op. cit. In particular the prodrugs are believed tobe metabolisable by CYPI Bl enzyme. Some of the compounds are new. Thepresent invention relates to the first therapeutic use of a broad rangeof compounds.

[0010] There is provided according to the first aspect of the inventionthe new use of a compound of the general formula I or a salt thereof inthe manufacture of a composition for use in a method of treatment bytherapy of an animal:

[0011] in which X is H;

[0012] Y is a leaving group

[0013] R¹ is —Ar, —NH₂, R⁸ or OR⁸;

[0014] R² and R⁴ are each independently selected from H, C₁₋₄ alkyl,—OH, C₁₋₄alkoxy, —CN, Cl, Br, I, —NO₂, —NH₂, —NHCOR⁹, —NHCOOR⁹, —COOH,—CONHR⁹ and —COOR⁹;

[0015] R³ is selected from H, C₁₋₄ alkyl, —OH, C₁₋₄ alkoxy, —CN, Cl, Br,I, —NO₂, —NH₂, —NHCOR⁹, —NHCOOR⁹, —COOH, —CONHR⁹, —COOR⁹ and COAr¹⁰;

[0016] R⁸ and, R⁹ are independently selected from C₁₋₄alkyl, optionallysubstituted phenyl, C₇₋₁₂-aralkyl, optionally substituted heteroaryl andligands;

[0017] Ar is selected from.

[0018] in which B is N or CR¹⁴;

[0019] Z is O, S—CH═CH— or NH;

[0020] the or each R¹¹ is selected from OH, C₁₋₄ alkoxy, C₁₋₄ alkyl,—NO₂, —NH₂, —NHR¹⁰, —NR¹⁰ ₂, —N⁺R¹⁰ ₃, —CN, Cl, Br, I, —NHCOR¹⁵, —COOH,—CONHR¹⁶, —NHCOOR¹⁶ and COOR¹⁶;

[0021] n is an integer in the range 0 to 4;

[0022] s the or each R¹⁰ is selected from C₁₋₄ alkyl, optionallysubstituted phenyl, C₇₋₁₂-aralkyl, optionally substituted heteroaryl andligands;

[0023] R¹² is H, —COAr¹, —CONH₂—COOH, —COR¹⁶ or —COOR¹⁶;

[0024] the or each R¹³ is selected from OH, C₁₋₄ alkoxy, C₁₋₄ alkyl,—NO₂, —NH₂, —NHR¹⁰, —NR¹⁰ ₂, —N⁺R¹⁰ ₃, —CN, Cl, Br, I, —NHCOR¹⁵, —COOH,—CONHR¹⁶, —NHCOOR¹⁶ and —COOR¹⁶;

[0025] m is 0, 1 or 2;

[0026] R¹⁴ is selected from OH, C₁₋₄ alkoxy, C₁₋₄ alkyl, —NO₂, —NH₂,—CN, Cl, Br, I, —NHCOR¹⁵, —COOH, —CONHR¹⁶, —NHCOOR¹⁶—COOR¹⁶ and H;

[0027] R¹⁵ is selected from C₁₋₄ alkyl, optionally substituted phenyl,optionally substituted heteroaryl, C₇₋₁₂ aralkyl, Ar¹ and ligands;

[0028] R¹⁶ is selected from C₁₋₄alkyl, optionally substituted phenyl,C₇₋₁₂-aralkyl, optionally substituted heteroaryl and ligands;

[0029] Ar¹⁰ is

[0030] in which x is 0, 1 or 2;

[0031] Ar¹ is selected from the same groups as Ar; provided that no morethan one group R¹¹ or R¹³ in any one ring includes a group Ar¹.

[0032] The animal which is treated is generally a human, although thecompounds may also have veternary use. The indication treated isgenerally cancer, including adenocarcinoma, leukemia, lymphoma,melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancersof the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix,gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver,lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivaryglands, skin, spleen, testis, thymus, thyroid, and uterus. The tumourmay, for instance, be defined s as a tumour expressing high levels ofCYP1B1.

[0033] In the invention a group Ar¹ is preferably

[0034] In the invention, the leaving group Y is, for instance, a groupwhich has utility as a leaving group in nucleophilic substitutionreactions. Suitable examples of such groups are —OCOOR⁵, —OCONHR⁶, Cl,Br, I, or —OSOOR⁷, in which R⁵, R⁶ and R⁷ are independently selectedfrom C₁₋₄alkyl, optionally substituted phenyl, C₇₋₁₂-aralkyl andoptionally substituted heteroaryl. Most preferably the leaving group isa halogen atom, preferably chlorine.

[0035] Optional substituents in phenyl, aralkyl and heteroaryl groupsare, for instance, C₁₋₄-alkyl, halogen, hydroxyl, C₁₋₄-alkoxy, —NH₂,—NHR¹⁰—, —NR¹⁰ ₂, —N⁺R¹⁰ ₃, —NO₂—, —CN, —COOH, —NHCOR⁵, —COOR¹⁵,—NHCOOR¹⁶ CONHR¹⁸ etc.

[0036] In the present invention the term ligand includes a group havingspecific targeting characteristics, useful for instance in antibody orgene-directed enzyme prodrug-type environments. A ligand may be anoligopeptide, biotin, avidin or streptavidin, a polymeric group, anoligonucleotide or a protein. Preferably it has specific bindingcharacteristics such as an antibody or fragment, an antigen, a sense oranti-sense oligo-nucleotide, or one of avidin, streptavidin and biotin,that is it is one component of a specific binding pair. Alternatively itmay be a group designed for passive targeting, such as a polymericgroup, or a group designed to prolong the stability or reduceimmunogenicity such as a hydrophilic group. U.S. Pat. No. 5,843,937discloses suitable ligands for conjugating to these types of actives andmethods for carrying out the conjugation.

[0037] In a pharmaceutically active compound R¹ is other than OR⁸. Ingeneral, for optimised DNA binding ability, the group R¹ in a compoundof the general formula I and IA is a group Ar and/or the group R³ is agroup Ar¹⁰. Often the group R¹ may include two aromatic groups joined toone another. In such compounds, one of the groups R₁₁ of the Ar group,or the group R¹², as the case may be, is a group Ar¹. Whilst for somecompounds it may be desirable for three or more such aromatic groups tobe linked, it is preferred that there is one group Ar and either onegroup Ar¹⁰, or, more preferably, one group Ar¹. Thus in a group Ar¹which is a pyrrolo-dihydroindole type of group, the group R¹² should beother than a group —COAr¹. In a group Ar¹ which is one of the othertypes of group there should either be no substituents R¹¹, or R¹³ as thecase may be, or, if there are any substituents, such substituents shouldnot include a group Ar¹.

[0038] According to one embodiment of the invention, the substituent Aris a group

[0039] In such groups Ar, B is preferably CR¹⁴. R¹⁴ is preferably H. Thedefinition of Z is preferably NH, although furan (Z is O) and thiophene(Z is 5) analogues had been generated for conjugation to DNA alkylatingunits and may have useful DNA binding characteristics. Similarly, in agroup Ar¹, the groups B and Z are selected amongst the same preferablegroups. Preferably n is at least 1 and one of the groups R¹¹ is—NHCOAr¹. In this embodiment Ar¹ is preferably a group

[0040] in which B and Z are the same as in Ar.

[0041] In another embodiment the substituent Ar is a group

[0042] Preferably R¹² in such a group Ar is a group —COAr¹ in which Ar¹preferably is the same type of group. Alternatively R¹² in such a groupis other than —COAr¹ and R³ is —COAr¹⁰.

[0043] In both groups Ar and Ar¹, m in the indole type group ispreferably zero.

[0044] In Ar and Ar¹, there may be several substituents R¹¹. Mostpreferably such substituents are selected amongst C₁₋₄-alkoxy groups.

[0045] In compounds of the formula I, the core indole ring of the DNAalkylating sub-unit is preferably unsubstituted in the benzene ring (R²is hydrogen), whilst the pyrrole ring may be unsubstituted (R³ and R⁴are both hydrogen, or one or both of them represents a group —COOR¹⁰, ora C₁₋₄-alkyl, preferably methyl).

[0046] In the compounds of the formula I, X is H. It is believed that,hydroxylation of the compound will occur in situ at the carbon atom towhich X is attached, thereby activating the compound enabling it to actas a DNA alkylating agent.

[0047] Many of the compounds of the general formula I and IA, as well asamine protected precursors thereof are believed to be novel compounds.According to a further aspect of the invention there is provided a newcompound of the general formula 11 or IIA or a salt thereof

[0048] in which R², R³ and R⁴ are as defined for formula. I and IAabove;

[0049] X¹ is H;

[0050] Y¹ is a leaving group;

[0051] R¹⁸ is H or an amine protecting group;

[0052] R¹⁷ is R⁸, —OR⁸—NH₂ or Ar²;

[0053] R⁸ is as defined above for formula I and IA;

[0054] Ar² is selected from

[0055] in which B^(t) is N or CR⁴⁰,

[0056] R⁴⁰ is selected from H, OH, C₁₋₄-alkoxy, C₁₋₄-alkyl, —NO₂, —NH₂,—CN, Cl, Br, I, —NHCOR²², —COOH, —CONHR²³, —NHCOOR²³ and —COOR²³.

[0057] Z¹ is O, S, —CH═CH— or NR¹⁸;

[0058] the or each R¹⁹ is selected from, OH, C₁₋₄ alkoxy C₁₋₄ alkyl,NO₂, —NHR¹⁸, —NHR²³, —NR²³ ₂, —N⁺R²³ ₃, —CN, Cl, Br, I, —NHCOR²², —COOH,—CONHR²³ and —COOR²³;

[0059] p is an integer in the range 0 to 4;

[0060] R²⁰ is H, —COAr³, —CONH₂, —COOH, —COR²³ or —COOR²³;

[0061] the or each R²¹ is selected from OH, C₁₋₄ alkoxy C₁₋₄ alkyl, NO₂,—NHR¹⁸, —NHR²³, —NR²³ ₂, —N⁺R²³ ₃, —CN, Cl, Br, I, —NHCOR²², —COOH,—CONHR²³ and —COOR²³;

[0062] q is 0, 1 or 2

[0063] R²² is selected from C₁₋₄ alkyl, optionally substituted phenyl,optionally substituted heteroalkyl, C₇₋₁₂ aralkyl, ligands and Ar³

[0064] R²³ is selected from C₁₋₄alkyl, optionally substituted phenyl,C₇₋₁₂-aralkyl and optionally substituted heteroaryl; and

[0065] Ar³ is selected from the same groups as Ar² provided that no morethan one R¹⁹ or R²¹ in any one ring includes a group Ar³.

[0066] Ar³ is preferably.

[0067] Compounds of the formula II or IIA, in which primary or secondaryamine nitrogen atoms are protected are generally deprotected beforebeing used in pharmaceutical compositions. Examples of amine protectinggroups are benzyl, benzyloxycarbonyl, tertiary butyloxycarbonyl (BOC),fluorenyl-N-methoxy-carbonyl (FMOC) and2-[biphenylyl-(4)]-propyl-2-oxycarbonyl. In particularly useful servicesof compounds of the general formula II and IIA R¹⁷ is —OR⁸ and R⁸ is anamine protecting group different to R⁸OCO—. In another preferredservices R¹⁷ is other than DR⁸. Where more than one such amine group isprotected in the molecule, the protecting groups may be the same ordifferent.

[0068] The present invention further provides pharmaceuticalcompositions comprising compounds of the formula I or IA or salts and apharmaceutically acceptable excipient. Pharmaceutical compositions maybe suitable for intramuscular, intraperitoneal, intrapulmonary, oral or,most preferably, intravenous administration. The compositions containsuitable matrixes, for example for controlled or delayed release. Thecompositions may be in the form of solutions, solids, for instancepowders, tablets or implants, and may comprise the compound of theformula I in solid or dissolved form. The compound may be incorporatedin a particulate drug delivery system, for instance in a liquidformulation. Specific examples of suitable excipients include lactose,sucrose, mannitol, and sorbitol; starch from corn, wheat, rice, potato,or other plants; cellulose, such as methyl cellulose,hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums,including arabic and tragacanth; and proteins, such as gelatin andcollagen. If desired, disintegrating or solubilizing agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, and alginicacid or a salt thereof, such as sodium alginate. Solid compositions maytake the form of powders and gels but are more conveniently of a formedtype, for example as tablets, cachets or capsules (including spansules).Alternative, more specialised types of formulation including liposomes,nanosomes and nanoparticles.

[0069] Compounds of the formula I and IA may be synthesised usingtechniques analogous to those summarised by Boger et al 1997, op. cit.It is convenient to form the DNA alkylating sub unit in one series ofsteps and to attach this through the nitrogen atom of thedihydro-pyrrole or tetrahydroquinoline, as the case may be, (C) ring tothe rest of the molecule. The DNA alkylating sub-unit may be conjugatedto DNA binding sub-units synthesised as described in Boger et al, 1997op. cit., for instance the PDE-1 and PDE-11 sub-units described in thatreference. The DNA binding subunits are the groups including Ar, Ar¹ andAr¹⁰.

[0070] According to a further aspect of the invention there is provideda new synthetic method in which a compound of the formula IV or IVA

[0071] in which X², R² and R⁴ are as defined above;

[0072] R³ is selected from the same groups as R³;

[0073] Y² is a leaving group or a hydroxyl or protected hydroxyl group;and

[0074] R²⁶ is an amine protecting group;

[0075] is reacted with a compound of the general formula V

R²⁷COY³  V

[0076] in which R²⁷ is selected from C₁₋₄-alkyl, optionally substitutedphenyl, C₇₋₁₂-aralkyl, optionally substituted heteroaryl and Ar⁴;

[0077] Ar⁴ is selected from

[0078] in which B² is N or CR³²;

[0079] Z² is O, S, —CH═CH— or NR³³;

[0080] the or each R²⁸ is selected from C₁₋₄-alkoxy, C₁₋₄-alkyl, NO₂,CN, Cl, Br, I, —NHR³³, —NR³⁶ ₂, —N+R³⁵ ₃—, —NHCOR³⁴, —COOH, —CONHR³⁵ and—COOR³⁵;

[0081] r is an integer in the range 0 to 4;

[0082] R²⁹ is an amine protecting group;

[0083] R³⁰ is an amine protecting group, —CONH₂, —COOH, —COR³⁵ or—COAr⁵;

[0084] the or each R³¹ is selected from C₁₋₄-alkoxy, C₁₋₄-alkyl, NO₂,CN, Cl, Br, I, —NHR³³, —NR³⁶ ₂, —N⁺R³⁶ ₃—, NHCOR³⁴, —COOH, —CONHR³⁵ and—COOR³⁵;

[0085] s is 0, 1 or 2;

[0086] R³² is selected from H, C₁₋₄-alkoxy, C₁₋₄-alkyl, NO₂, CN, Cl, Br,I, NHCOR³⁴, —COOH, —CONHR^(35,)—NHCOOR³⁵ and COOR³⁵;

[0087] the or each R³³ is an amine protecting group;

[0088] R³⁴ is selected from Ar⁵, C₁₋₄-alkyl, optionally substitutedphenyl, C₇₋₁₂-aralkyl, optionally substituted heteroaryl and ligands;

[0089] R³⁵ is selected from C₁₋₄-alkyl, optionally substituted phenyl,C₇₋₁₂-aralkyl, optionally substituted heteroaryl and ligands;

[0090] each R³⁶ is selected from C₁₋₄-alkyl, optionally substitutedphenyl, C₇₋₁₂-aralkyl, optionally substituted heteroaryl and H

[0091] Ar⁵ is selected from the same groups as Ar⁴; and

[0092] Y³ is a leaving group, provided that no more than one R²⁸ or R³¹in any one ring includes a group Ar⁵.

[0093] Ar⁵ is preferably

[0094] Y³ is, for instance, selected amongst the preferred leavinggroups listed above for Y. Most suitably the definition of Y³ is Cl.Alternatively, the group Y³ may be OH. In this case, it may be necessaryto include a coupling agent to assist in the coupling reaction.

[0095] The reaction between the compound of the general formula IV orIVA and the carboxylic acid or derivative of the general formula V iscarried out under conditions allowing such coupling to take place. Suchconditions are similar to those generally used for formation of peptidebonds, for instance as used in peptide synthetic methods.

[0096] Y² is a hydroxy group or a leaving group, which may be the sameas Y or may be converted to Y in a subsequent step.

[0097] Where, in the product, R³ is a different group to R³⁷, the R³⁷group is subsequently derivatised to generate the desired group R³. Thisis often the case where, for instance, R³ is a group Ar¹⁰ or —CONHR⁹. Toproduce compounds of that type the group R³⁷ is, for instance, —COOH or—COOR¹⁰ and is reacted, optionally after hydrolysis/deprotection of agroup —COOR¹⁰, with an appropriate amine compound (R⁹NH₂ or Ar¹⁰H),optionally in the presence of coupling agents, to produce the amidelinked compound.

[0098] After the coupling process, it may be desirable to deprotect oneor more of the protected amine groups. If further reaction, for-instancewith other derivatising agents such as glycosyl compounds, peptides,polymers etc is desired through any such amine groups, it may bedesirable to deprotect only those to which subsequent reaction to totake place, whilst retaining the other amine groups in a protected form.Selection of suitable amine protecting groups and protection anddeprotection protocols may be made using techniques commonly utilised inpeptide chemistry.

[0099] It is believed that some of the intermediates of the generalformula IV or IVA may be novel compounds. According to a further aspectof the invention, there is provided a novel compound of the generalformula III or IIIA

[0100] in which R² and R⁴ are as defined for formula I and IA above;

[0101] R³⁸ is selected from the same groups as R³;

[0102] X² is H;

[0103] Y² is a leaving group or a hydroxyl or protected hydroxyl group;and

[0104] R²⁴ and R²⁵ are each H or an amine protecting group.

[0105] In compounds of the general formula III and IIIA, in the compoundready for reaction with a carboxylic acid derivative, for instance ofthe general formula V, R²⁴ is H, whilst R²⁵ should be an amineprotecting group. Precursors for such compounds may have both ringnitrogen atoms in protected form, that is in which R²⁵ and R²⁴ representprotecting groups. In such compounds, since it is desired for thecompound to be capable of derivatisation at just one of the nitrogenatoms, preferably R²⁴ and R²⁵ represent different protecting groups.

[0106] In compounds of the formula III and IIIA, the group Y² may beselected amongst those defined above for leaving group Y. The nature ofthe group Y² should be selected having regard to the nature of thereagent with which the compound of the formula IV or IVA, as the casemay be, is to react in a subsequent step. Suitable examples of leavinggroup Y² are selected from those listed above for Y.

[0107] The compound of the formula III may be prepared in a preliminarystep using as the starting material an aniline compound having a leavinggroup substituent Y⁴ at the carbon atom ortho to the amine groupsubstituent, and an N-substituent which is a trans 2-propen-1-yl group—CH₂CH═CHY, in which Y⁵ is hydrogen or a group which is the same as Y²or may be converted to into Y² in a subsequent step in which the anilinederivative is reacted under cyclisation conditions, to form adihydropyrrole ring. Preferably in the cyclisation reaction a halogenY⁵(═Y²) substituent is retained. The group Y⁴ should be a radicalleaving group, such as halogen, preferably I or Br. Suitable radicalsfor carrying out the cyclisation reaction where r is hydrogen arenitroxy compounds such as 2,2,6,6-tetramethylpiperidinyloxy (TEMPO).Where Y is a radical leaving group (gY²) the reaction may be carried outin the presence of a radical derived from azoisobutyronitrile (AIBN). Inthis step r does not leave. Suitable catalysts for a radical cyclisationstep are tin hydride compounds such as tributyl tin hydride. Thissynthetic pathway is illustrated in Examples 1 and 3.

[0108] The compound of the general formula IIIA may be formed bycyclisation of an aniline compound having a radical leaving group Y⁴substituent ortho to the amine group and an N-substituent which is a2-propen-1-yl group, preferably a trialkyl tin radical, undercyclisation conditions to form an intermediate dihydroquinonone. Thecyclisation reaction is conducted in the presence of suitable catalystswhich are, for instance, palladium complexes such as tetrakis(triphenylphosphine) palladium (0), bis(triphenyl phosphine) palladium(II) chloride or palladium (II) acetate. The dihydroquinonineintermediate is oxidised to form a further intermediate which is anepoxide, for instance using a peroxide reagent. The epoxide intermediateis reduced using a suitable selective reducing agent such as a dialkylaluminium hydride to produce the corresponding alcohol which issubsequently halogenated, for instance using carbontetrachloride/triphenyl phosphine. This reaction is illustrated inExamples 2 and 4.

[0109] The starting compound for such reactions may be represented bythe general formula VI

[0110] in which R², R⁴, R²⁶, and X² are the same as in the compound ofthe formula IV;

[0111] R³⁹ is selected from the same groups as R³;

[0112] R⁴⁰ is an amine protecting group different from R²⁶,

[0113] one of Z¹ and Z² is Y⁵ and the other is H;

[0114] Y⁵ hydrogen, or is a leaving group which is the same as ordifferent to Y²; and

[0115] Y⁴ is a radical leaving group.

[0116] Y⁴ is preferably selected from Cl, Br and I.

[0117] The compound of the general formula VI may be produced byalkylation of the sodium salt of the corresponding amiline derivativewith a cis or trans-1,3-dihalo prop-2-ene compound. The cis startingmaterial produces a compound of the general formula IV in which Z² isY⁵, the trans stating material a product VI in which Z¹ is Y⁵. An allylreagent produces a compound VI in which Y⁵ is hydrogen,

[0118] The carboxylic acid derivative of the general formula V may besynthesised using the methods generally described in Boger et al, 1997 Sop.cit, for instance PDE-I and PDE-II may be synthesised using theUmezawa synthesis, the Rees-Moody synthesis, the Magnus synthesis, theCava-Rawal synthesis, the Boger-Coleman synthesis, the Sundbergsynthesis, the Martin synthesis, the Tojo synthesis. Indole-2-carboxylicacid is commercially available. Other analogues of the DNA bindingsub-units of the duocarmycins, and reactive carboxylic acid derivativesthereof are described by Boger et al, op.cit. and in U.S. Pat. No.5,843,937.

[0119] Two specific examples of compounds of the general formula I andII are

[0120] Two specific examples of a compound of the general formula IA andIIA are:

[0121] Other examples are ethyl rather than methyl esters of compoundsVII and X.

[0122] The present invention relates to the creation of a range ofprodrugs that have little or no cytotoxic effects when in their normalstate, but are highly cytotoxic (i.e. have a substantially increasedcytotoxicity) when activated by oxidation or hydroxylation by CYPenzymes. This provides for a self-targeting drug delivery system inwhich a non cytotoxic (or negligibly cytotoxic) compound can beadministered to a patient, for example in a systemic manner, thecompound then being activated at the site of the tumour cells(intratumoural activation) to form a highly cytotoxic compound whichacts to kill the tumour cells. The fact that the CYP isoforms are notexpressed by normal cells mean that the activation of the compound onlyoccurs at the site of the tumour cells and therefore only tumour cellsare affected, thus providing a self-targeting system.

[0123] The prodrugs of the present invention have the distinct advantageof being useful in the treatment of tumours at any site in the body,meaning that even tumours that have undergone metastasis (which arenormally not susceptible to site specific therapies) may be treated.

[0124] The prodrug may be an antitumour prodrug. Examples of tumoursinclude cancers (malignant neoplasms) as well as other neoplasms e.g.innocent tumours. The prodrug may be activated by hydroxylation byisoforms of cytochrome P450's.

[0125] In a variation of the normal procedure which relies upon CYPexpression within tumour cells to effect selective hydroxylation andhence activation of the prodrugs, the selectivity between tumour tissueand normal tissue can be enhanced in a two part procedure. Thus (a)infecting tumor cells with a viral vector carrying a cytochrome P450gene and a cytochrome P450 reductase gene, wherein expression ofcytochrome P450 gene and cytochrome P450 reductase gene by tumor cellsenables the enzymatic conversion of a chemotherapeutic agent to itscytotoxic form within the tumor, whereby the tumor cells becomeselectively sensitized to the prodrug chemotherapeutic agent (b)contacting tumor cells with the prodrug chemotherapeutic agent wherebytumor cells are selectively killed.

[0126] These prodrugs are pyrrolodihydroindole (general formula I) orpyrrolo tetrahydroquinoline (general formula IA) derivatives. Theirspecific use as antitumour prodrugs has not been previously suggested ordisclosed, nor has the suggestion that they are prodrugs having anactivated hydroxylated form. Where compounds of formula (I) have beenpreviously identified and made, they have not been identified asanti-tumour agents due to their poor (or negligible) cytotoxicity. Thusthe intratumoural hydroxylation of the prodrugs of the present inventionprovides-them with a surprising and unexpected efficacy.

[0127] Hydroxylated forms of the prodrugs are potent DNA alkylatingagents that bind in the minor groove of DNA and alkylate the purinebases at the N3 position. As such, they are potent cytotoxic agentswhose exact biological mechanism of action is unknown but involves thedisruption of template and other functions of DNA. General inhibition oftemplate function of DNA will affect and be generally cytotoxic to alldividing cells in the body and lead to unacceptable side effects in atherapeutic setting. However, the targetted production of hydroxylatedforms only in tumour cells that overexpress particular isoforms ofcytochrome P450's will lead to a specific cytotoxic effect only in thosecells. The non-hydroxylated forms are essentially non-toxic to allcells.

[0128] The following examples illustrate the invention:

EXAMPLE 1

[0129] The synthesis of one compound of the general formula I is carriedout according to the following reaction scheme.

[0130] 1.1 1-Benzoyl-5-nitroindole

[0131] 5-nitroindole (100 mg, 0.62 mmol) in CH₂Cl₂ (1 mL) is treatedwith benzoyl is chloride (86 mg, 0.62 mmol, 1 equiv.) and4-dimethylaminopyridine (74 mg, 0.62 mmol, 1 equiv.). The mixture isstirred at room temperature for 1 h, diluted with CH₂Cl₂ (10 mL), washedwith HCl (1M, 2×10 mL) and water (1×10 mL), dried (MgSO₄) andconcentrated. Chromatography (Silica gel, 2×15 cm, 10% ethylacetate/hexanes) gives the product.

[0132] 1.2 2-Amino-1-benzoylindole

[0133] 1-Benzoyl-5-nitroindole (100 mg, 0.38 mmol) in ethyl acetate (2mL) is treated with 10% Pd/C (10 mg) and stirred under an atmosphere ofhydrogen at room temperature for 4 h. The resulting solution is filteredthrough celite and concentrated. Chromatography (Silica gel, 2×15 cm,10% ethyl acetate/hexanes) gives the product.

[0134] 1.3 5-Amino-1-benzoyl-4-iodoindole

[0135] 5-Amino-1-benzoylindole (100 mg, 0.42 mmol) in tetrahydrofuran(THF) (1 mL) is treated with N-iodosuccinimide (103 mg, 0.46 mmol, 1.1equiv) and 4-toluenesulfonic acid (16 mg, 0.08 mmol. 0.2 equiv.) andstirred at room temperature (RT) for 16 hours. The solution isconcentrated and redissolved in ethyl acetate (10 mL). The organic layeris washed with water (1×10 is mL), 1M HCl (2×10 mL) and water (1×10 mL),dried (MgSO₄) and concentrated. Chromatography (Silica gel, 2×15 cm, 10%ethyl acetate/hexanes) gives the product.

[0136] 1.4 1-Benzoyl-5-((tert-butyloxy)carbonyl)amino-4-iodoindole

[0137] 5-Amino-1-benzoyl4-iodoindole (100 mg, 0.28 mmol) is stirred inCH₂Cl₂ (2 mL) and treated with di-tert-butyl-dicarbonate (89 mg, 0.41mmol, 1.5 equiv), triethylamine (57 μL, 0.41 mmol, 1.5 equiv) and4-dimethylaminopyridine (4 mg, 0.028 mmol, 0.1 equiv). After 16 h at RT,the solvents are removed under reduced pressure. Chromatography (Silicagel, 2×15 cm, 10% ethyl acetate/hexanes) gives the product.

[0138] 1.51-Benzoyl-5-[N-(3-Chloro-2-propen-1-yl)-N-((tert-butyloxy)carbonyl)]amino-4-iodoindole

[0139] 1-Benzoyl-5-(tert-butyloxycarbonyl)amino-4-iodoindole (100 mg,0.22 mmol) was stirred in DMF (1 mL) and sodium hydride (26 mg, 0.66mmol, 60% dispersion in oil, 3 equiv.) is added. After 15 min, thesuspension is treated with E/Z-1,3-dichloropropene (61 μL, 0.66 mmol, 3equiv) and the resulting solution was stirred at RT for 16 h. Thesolution is concentrated and water (10 mL) is added. The aqueoussolution was extracted with ethyl acetate (3×10 mL), the organic layerscombined, dried and concentrated. The product was obtained afterchromatography (Silica gel, 2×15 cm, 10% ethyl acetate/hexanes).

[0140] 1.61-(Chloromethyl)-6-benzoyl-3-((tert-butyloxy)carbonyl)-1,2-dihydro-3H-pyrrolo[3,2-e]indole

[0141]1-Benzoyl-5-[N-(3-Chloro-2-propen-1-yl)-N-((tert-butyloxy)carbonyl)]amino-4-iodoindole(100 mg, 0.19 mmol), poly(methylhydrosiloxane) (200 μL),bis(tributyltin) oxide (19 μL, 0.04 mmol, 0.2 equiv) and azoisobutyronitrile (AIBN) (6 mg, 0.04 mmol, 0.2 equiv) were stirred intoluene (2 mL) at 80° C. under N₂ for 4 h. The solvent is then removedin vacuo. Chromatography (Silica gel, 2×15 cm, 10% ethylacetate/hexanes) gives the product.

[0142] 1.7 5-Methoxyindole extended agent.1-(chloromethyl)₆-benzoyl-3-((5-methoxy-1H-indol-2-yl)carbonyl)-1,2-dihydro-3H-pyrrolo[3,2-e]indole

[0143]1-(Chloromethyl)-6-benzoyl-3-((tert-butyloxy)carbonyl)-1,2-dihydro-3H-pyrrolo[3,2-e]indole(100 mg, 0.24 mmol) is treated with a solution of hydrochloric acid inethyl acetate (4M, 500 μL). After 30 min, the solvent is concentratedand dimethyl formamide (DMF) (1 mL) is added. The solution is treatedwith 1-[(3-dimethylamino)propyl]-3-ethyl carbodimide (EDC) (140 mg, 0.73mmol) and 5-methoxyindole-2-carboxylic acid (140 mg, 0.73 mmol). After16 h, the solvent is removed under reduced pressure. Chromatography(Silica gel, 2×15 cm, 10% ethyl acetate/hexanes) gives the product. The6-benzoyl protecting group may be removed by sodium methoxide inmethanol, followed by removal of the solvent.

EXAMPLE 2

[0144] The following example illustrates the synthesis of a compound ofthe general formula 1A in which R¹ is OR⁸ and R⁸ is tBu. It is suitablefor extending by a step analogous to step 1.7 above, to form a furthercompound of the formula IA in which R¹ is Ar, and optionallysubsequently deprotected at the nitrogen atom of the indole ring.

[0145] z2.11-Benzoyl-5-[N-(3-(tributylstannyl)-2-propen-1-yl)-N-((tert-butyloxy)carbonyl)]amino-4-iodoindole

[0146] 1-benzoyl-5-(tert-butyloxycarbonyl)amino-4-iodoindole (100 mg,0.22 mmol) is stirred in DMF (1 mL) and sodium hydride (26 mg, 0.66mmol, 60% dispersion in oil, 3 equiv.) is added. After 15 min, thesuspension is treated with E/Z-1-tributylstannyl-3-bromopropene (270 mg,0.66 mmol, 3 equiv) (Boger, D. L.; McKie, J. A.; Boyce, C. W. Synlett1997, 515-516) and the resulting solution is stirred at RT for 16 h. Thesolution was concentrated and water (10 mL) is added. The aqueoussolution is extracted with ethyl acetate (3×10 mL), the organic layerscombined, dried and concentrated. The product was obtained afterchromatography (Silica gel, 2 15 cm, 10% ethyl acetate/hexanes)

[0147] 2.21,2-Dihydro-1-((tert-butyloxy)carbonyl)-5,6-(9-benzoylpyrrolo)quinoline.

[0148]1-Benzoyl-5-[N-(3-(tributylstannyl)-2-propen-1-yl)-N-((tert-butyloxy)carbonyl)]amino-4-iodoindole(100 mg, 0.12 mmol) and tetrakis(triphenylphosphine)palladium(0) (32 mg,0.2 equiv) are stirred in toluene (2 mL) at 50° C. under N₂ for 2 h. Thesolvent is then removed in vacuo. Chromatography (Silica gel, 2×15 cm,10% ethyl acetate/hexanes) gives the product.

[0149] 2.33,4-Epoxy-1-((tert-butyloxy)carbonyl)-1,2,3,4-tetrahydro-5,6-(9-benzoylpyrrolo)quinoline.

[0150]1,2-dihydro-1-((tert-butyloxy)carbonyl)-5,6-(9-benzoylpyrrolo)quinoline.(100 mg, 0.27 mmol) and 3-chloroperoxy benzoic acid (68 mg, 0.40 mmol,1.5 equiv) were stirred in CH₂Cl₂ (2 mL) at −78° C. to −30° C. under N₂for 2 h. The solvent is then removed in vacuo. Chromatography (Silicagel, 2×15 cm, 10% ethyl acetate/hexanes) gives the product.

[0151] 2.44-Hydroxy-1-((tert-butyloxy)carbonyl)-1,2,3,4-tetrahydro-5,6-(9-benzoyl)pyrroloquinoline.

[0152]3,4-epoxy-1-((tert-butyloxy)carbonyl)-1,2,3,4-tetrahydro-5,6-(9-benzoylpyrrolo)quinoline(100 mg, 0.26 mmol) was treated with disobutyl aluminium hydride(Dibal-H) (55 mg, 0.39 mmol, 1.5 equiv) in THF (2 mL),at −78° C. to −30°C. under N₂. After 1 h, the reaction is quenched by the addition ofwater (2 mL) and the resulting solution is extracted with ethyl acetate(3×10 mL), the organic layers combined, dried and concentrated. Thesolvent is removed in vacuo. Chromatography (Silica gel, 2×15 cm, 10%ethyl acetate/hexanes) gives the product.

[0153] 2.54-Chloro-1-((tert-butyloxy)carbonyl)-1,2,3,4-tetrahydro-5,6-(9-benzoyl)pyrroloquinoline

[0154]4-hydroxy-1-((tert-butyloxy)carbonyl)-1,2,3,4-tetrahydro-5,6-(9-benzoyl)pyrroloquinoline(100 mg, 0.26 mmol) in CH₂Cl₂ (2 mL) is treated with a prepared solutionof PPh₃ (137 mg, 0.52 mmol, 2 equiv) and CCl₄ (200 mL) in CH₂Cl₂ (2 mL)at RT. After 4 h, the solvent is removed in vacuo. Chromatography(Silica gel, 2×15 cm, 10% ethyl acetate/hexanes) gives the product.

EXAMPLE 3

[0155]

[0156] 3.1 Ethyl 1-benzoyl-5-nitroindole-2-carboxylate (3.2)

[0157] Ethyl 5-nitroindole-2-carboxylate (3.1) (1.5 g, 6.41 mmol) inCH₂Cl₂ (30 ml) was treated with benzoyl chloride (1.19 ml, 10.26 mmol),Et₃N (891 μl, 6.41 mmol) and DMAP (783 mg, 6.41 mmol). The mixture wasstirred for 16 h. 10% NaHCO₃ (10 ml) and CH₂Cl₂ (10 ml) were added andthe organic layer was separated. The aqueous layer was extracted withCH₂Cl₂ (3×5 mL). The combined organic layers were washed with H₂O (10ml), 5% HCl (10 ml) and H₂O (10 ml). The solution was dried (MgSO₄) andconcentrated. The 10 residue was crystalised from EtOAc/Hex 1:9 toafford 1.85 g (87%) of 3.2 as a yellow powder: ¹H NMR (250 MHz, CDCl₃) δppm 8.66 (d, 1H), 8.25 (d, 1H), 7.80 (d,1H), 7.6-7.72 (m, 3H), 7.48 (m,3H), 4.00 (q, 2H), 1.10 (t, 3H). FABMS (NBA/NaI) m/z 339 (M+H⁺ expected339)

[0158] 3.2 Ethyl 5-amino-1-benzoylindole-2-carboxylate (3.3).

[0159] A solution of 3.2 (1.86 g, 5.5 mmol) and 10% Pd/C (440 mg) in dryTHF (30 ml) was stirred under H₂ for 16 hrs. The resulting mixture wasfiltered through celite which was washed with EtOAc (40 ml) and thefiltrate was concentrated. The residue was purified by chromatography(SiO₂, 0 to 40% EtOAc in hexanes) to afford 3.3 (1.63 g, 96%) as abright yellow oil. ¹H NMR (250 MHz, CDCl₃) δ ppm 7.40-7.72 (m, 6H), 7.18(s,1H), 6.92 (d, 1H), 6.82 (dd, 1H), 3.92 (q, 2H), 3.68 (br s, 2H), 1.06(t, 3H); FABMS: (NBA/NaI) m/z 308 (M+H⁺ expected 308).

[0160] 3.3 Ethyl 5-amino-1-benzoyl-4-iodoindole-2-carboxylate (3.4)

[0161] 5-amino-1-benzoylindole (1.63 g, 5.29 mmol) in THF (75 mL) wastreated with N-iodosuccinimide (1.89 g, 8.46 mmol) and 4-toluenesulfonicacid (364 mg, 2.12 mmol) and stirred at RT for 16 hours. The solutionwas concentrated and redissolved in ethyl acetate (100 mL). The organiclayer was washed with water (1×100 mL), 1 M HCl (2×100 mL) and water(1×100 mL), dried (MgSO₄) and concentrated. Chromatography (SiO₂, 10%ethyl acetate/hexanes) gave the product (1.17 g, 51%) as a bright yellowsolid. ¹H NMR (250 MHz, CDCl₃) δ ppm 7.30-7.70 (m, 6H), 7.30 (s, 1 H),6.80 (d, 1 H), 4.05 (s, 2 H), 3.85 (q, 2 H), 1.0 (t, 3H) FABMS (NBA/NaI)m/z 434 (M+H⁺ expected 434),457 (M+Na⁺ expected 457).

[0162] 3.4 Ethyl1-benzoyl-5-(N-(tert-butyloxycarbonyl)-4-iodoindole-2-carboxylate (3.5)

[0163] A mixture of 3.4 (1.17 g, 2.70 mmol), (Boc)₂O (9.40 g, 43 mmol)and Et₃N (375 μL, 2.70 mmol) in dioxan (100 mL) was heated to 100° C.under N₂ for 48 h. Upon completion, the solution was cooled,concentrated and purified by flash chromatography (SiO₂, 0-20% EtOAc inhexane) to afford 3.5 (1.3 g, 90%) as a yellow oil. FABMS (NBA.NaI) 535(M+H⁺ expected 535).

[0164] 3.5 Ethyl1-benzoyl-5-[N-(3-chloro-2-propen-1-yl)-N-((tert-butyloxy)carbonyl)]amino-4-iodoindole(3.6)

[0165] 1-benzoyl-5-(tert-butyloxycarbonyl)amino-4-iodoindole (100 mg,0.22 mmol) was stirred in DMF (1 mL) and sodium hydride (26 mg, 0.66mmol, 60% s dispersion in oil, 3 equiv.) was added. After 15 min, thesuspension was treated with E/Z-1,3-dichloropropene (61 μL, 0.66 mmol, 3equiv) and the resulting solution was stirred at RT for 16 h. Thesolution was concentrated and water (10 mL) was added. The aqueoussolution was extracted with ethyl acetate (3′10 mL), the organic layerscombined, dried and concentrated. The (3.6) product was obtained afterchromatography (SiO₂, 10% ethyl acetate/hexanes) as a yellow oil (125mg, 94%). FABMS (NBA/NaI) m/z 609 (M+H⁺ expected 609).

[0166] 3.6 Ethyl6-benzoyl-1-(chloromethyl)-3-((tert-butyloxy)carbonyl)-1,2-dihydro-3H-pyrrolo[3,2-e]indole-7-carboxylate(3.7)

[0167] Compound 3.6 (100 mg, 0.19 mmol), and AIBN (6 mg, 0.04 mmol, 0.2equiv) were stirred in toluene (2 mL) at 80° C. under N₂. Bu₃SnH (51 μL,0.19 mmol) was added in 4 portions over 1 h. The solvent was thenremoved in vacuo. Chromatography (SiO₂ 10% ethyl acetate/hexanes) gavethe product (3.9). (72 mg, 78%) as an oil. FABMS (NaI/NBA) m/z 483(M+H+expected 483).

[0168] 3.7 Ethyl1-(chloromethyl)-3-((5-methoxy-1H-indol-2-yl)carbonyl)-1,2-dihydro-3H-pyrrolo[3,2-e]indole-7-carboxylate(3.8)

[0169] Compound 3.7(100 mg, 0.21 mmol) was treated with a solution ofhydrochloric acid in ethyl acetate (4M, 500 μL). After 30 min, thesolvent was concentrated and DMF (1 mL) was added. The solution wastreated with EDC (120 mg, 0.63 mmol) and 5-methoxyindole-2-carboxylicacid (120 mg, 0.63 mmol). After 16 h, the solvent was removed underreduced pressure and the residue (the 6N-benzoyl protected precursor ofcompound 3.8) was dissolved in CH₃OH (1 mL). A solution of NaOCH₃ inCH₃OH (2M, 100 μL) was then added and the solution stirred for 10minutes. The solvent was removed and chromatography (SiO₂, 10% ethylacetate/hexanes) gave the product (3.8) (100 mg, 86%). FABMS (NBA/NaI)mfz 557 (M+H⁺ expected 557).

EXAMPLE 4

[0170]

[0171] 4.1 Ethyl1-Benzoyl-5-[N-(3-(tributylstannyl)-2-propen-1-yl)-N-((tert-butyloxy)carbonyl)]amino-4-iodoindole-7-carboxylate(4.1)

[0172] Ethyl 1-benzoyl-5-(tert-butyloxycarbonyl)amino-4-iodoindole (3.5,synthesised as described in Example 3.1-3.4) (100 mg, 0.18 mmol) wasstirred in DMF (1 mL) and sodium hydride (21 mg, 0.54 mmol, 60%dispersion in oil, 3 equiv.) was added. After 15 min, the suspension wastreated with E/Z-1-tributylstannyl-3-bromopropene (221 mg, 0.54 mmol, 3equiv) and the resulting solution was stirred at RT for 16 h. Thesolution was concentrated and water (10 mL) was added. The aqueoussolution was extracted with ethyl acetate (3×10 mL), the organic layerscombined, dried and concentrated. The product was obtained afterchromatography (SiO₂, 10% ethyl acetate/hexanes) as a colourless solid(132 mg, 92%). FABMS (NBA/NaI) m/z 792 (M+H⁺ expected 792).

[0173] 4.25,6-(9-Benzoyl-8-(ethyloxy)carbonylpyrrolo)-1-((tert-butyloxy)carbonyl)-2,4-dihydroquinoline(4.2)

[0174] Compound 4.1 (100 mg, 0.12 mmol) andtetrakis(triphenylphosphine)palladium(0) (32 mg, 0.2 equiv) were stirredin toluene (2 mL) at 50° C. under N₂ for 2 h. The solvent was thenremoved in vacuo. Chromatography (SiO₂, 10% ethyl acetate/hexanes) gavethe product (50 mg, 94%) as a yellow oil. FABMS (NBA/NaI) m/z 447 (M+H⁺expected 447).

[0175] 4.35,6-(9-benzoyl-8-(ethyloxy)carbonylpyrrolo)-1-((tert-butyloxy)carbonyl)-3,4-epoxy—1,2,3,4-tetrahydroquinoline(4.3)

[0176] Compound 4.2 (100 mg, 0.22 mmol) and MCPBA (57 mg, 0.33 mmol, 1.5equiv) were stirred in CH₂Cl₂ (2 mL) at −30° C. under N₂ for 2 h. Thesolvent was then removed in vacuo. Chromatography (SiO₂, 10% ethylacetate/hexanes) gave the product (70 mg, 69%) as an oil. FABMS(NBA/NaI) m/z 463 (M+H⁺ expected 463).

[0177] 4.45,6-(9-benzoyl-8-(ethyloxy)carbonylpyrrolo)-1-((tert-butyloxy)carbonyl)-4-hydroxy-1,2,3,4-tetrahydroquinoline(4.4)

[0178] Compound 4.3 (100 mg, 0.22 mmol) was treated with Dibal-H (46 mg,0.33 mmol, 1.5 equiv) in THF (2 mL), at −30° C. under N₂. After 1 h, thereaction was quenched by the addition of water (2 mL) and the resultingsolution was extracted with ethyl acetate (3×10 mL), the organic layerscombined, dried and concentrated. The solvent was removed in vacuo.Chromatography (SiO₂, 10% ethyl acetate/hexanes) gave the alcohol (85mg, 83%). FABMS (NBA/NaI) m/z 465 (M+H⁺ expected 465).

[0179] 4.55,6-(9-benzoyl-8-(ethyloxy)carbonylpyrrolo)-1-((tert-butyloxy)carbonyl)-4-chloro-1,2,3,4-tetrahydroquinoline(4.5)

[0180] Compound 4.4 (100 mg, 0.22 mmol) in CH₂Cl₂ (2 mL) was treatedwith a prepared solution of PPh₃ (116 mg, 0.44 mmol, 2 equiv) and CCl₄(200 μL) in CH₂Cl₂ (2 mL) at RT. After 24 h, the solvent was removed invacuo. Chromatography (SiO₂, 10% ethyl acetate/hexanes) gave the targetcompound as an oil (95 mg, 90%). FABMS (NBA/NaI) m/z 484 (M+H⁺ expected484). The compound may be deprotected by removal of the tBOC group, aDNA-binding sub-unit conjugated to the nitrogen atom of the tCtrahydroquinoline ring and the indole nitrogen subsequently deprotected bysteps analogous to those of Example 3.7.

EXAMPLE 5 Biological Testing of Ethyl1-(chloromethyl)-3-((5-methoxy-1H-indol-2-yl)carbonyl)-1,2-dihydro-3H-pyrrolo[3,2-e]indole-7-carboxylate(3.8)

[0181] Materials and Methods

[0182] 5.1 Incubation Mixtures of Compound and Microsomes

[0183] Test compound (synthesised in example 3) activation by CYPenzymes was carried out using NADPH supplemented rat liver microsomes;Incubation mixtures comprised microsomal protein (1 mg/ml),reduced-nicotinamide adenine dinucleotide phosphate (NADPH, 10 mM) andphosphate buffer (pH7.4, 100 mM). Test compound (0.01-100 μM finalconcentration) in DMSO (20 μl) was added to the microsomal incubationmixtures (0.5 ml) and incubated for 60 min at 37C. Control incubatescontained test compound and microsomal incubation mixture terminated at0 time. All incubations were terminated by addition of an equal volumeof ice-cold acetonitrile and microfuged for 3 min. Aliquots of thesupernatant were added to cells in culture.

[0184] 5.2 Cell Culture Based Cytotoxicity Measurement

[0185] Chinese Hamster Ovary (CHO) cell were grown in MEM supplementedwith. 10% dialysed FBS and G418 (400 μg/ml). All cells were seeded at aninitial density of 1000 cells/well in 96-well-plates, incubation at 37°C. for 24 hours. Aliquots (0.1 ml) of the testcompound/microsomal/acetonitrile supernatnant was then added to the CHOcells. Cells were then incubated for 24 hours at 37° C., 5% CO₂. Afterthis time period MTT (50 μl; 2 mg/ml stock solution) was added to eachwell and cells were incubated for a further 4 hours. During this timeperiod MTT, a hydrogen acceptor tetrazolium salt, is reduced to formazandye by mitochondrial dehydrogenase of viable cells. The media wasaspirated from cells and DMSO (100 μl/well) added to solubilise thecoloured formazan dye. Absorbance of the formazan dye in the96-well-plates was then determined at 550 nm. The effect of microsomalactivation by the test compound on the arrest of CHO cell growth couldbe determined by comparing the IC₅₀ (concentration that inhibited cellgrowth by 50%) with and without microsomal incubation. Results CHO IC50(μM) compound +activation −activation AF 3.8 0.06 ± 0.02 4.3 ± 0.4171.7*

[0186] Effect of compound 3.8 and its metabolism (activation) product onthe survival of Chinese hamster ovary cells in culture. Cells wereincubated for 24 hours with supernatants from reaction mixtures ofcompound 3.8 with NADPH fortified rat liver microsomes. IC₅₀ representsthe concentration of drug required to inhibit cell growth by 50%. Valuesare expressed as the mean±sd for three experiments. See methods for fulldetails of metabolism. AF=activity factor i.e. the ratio of IC₅₀cytotoxicity values obtained for ±compound 3.8 activation.

[0187] represents significance at p>0.05.

1. Use of a compound of the general formula I or IA or a salt thereof inthe manufacture of a composition for use in a method of treatment bytherapy of an animal:

in which X is H; Y is a leaving group R¹ is —Ar, NH₂, R⁸ or —OR⁸; R² andR⁴ are each independently selected from H, C₁₋₄ alkyl, —OH, C₁₋₄ alkoxy,—CN, Cl, Br, I, —NO₂, —NH₂, —NHCOR⁹, —COOH, —CONHR⁹, —NHCOOR⁹, —COOR⁹and COAr¹⁰; R³ is selected from H, C₁₋₄ alkyl, —OH, C₁₋₄ alkoxy, —CN,Cl, Br, I, —NO₂, —NH₂, —NHCOR⁹, —COOH, —CONHR⁹, —NHCOOR⁹ and —COOR⁹; R⁸and R⁹ are independently selected from C₁₋₄alkyl, optionally substitutedphenyl, C₇₋₁₂-aralkyl and optionally substituted heteroaryl and ligands;Ar is selected from

in which B is N or CR¹⁴; Z is O, S —CH═CH— or NH; the or each R¹¹ isselected from —OH, C₁₋₄ alkoxy, C₁₋₄ alkyl, —NO₂, —NH₂, —NHR¹⁰, —NR¹⁰ ₂,—N⁺R¹⁰ ₃, —CN, Cl, Br, I, —NHCOR¹⁵ ₃—COOH, —CONHR¹⁶, —NHCOOR¹⁶ andCOOR¹⁶; n is an integer in the range 0 to 4; the or each of R¹⁰ isselected from C₁₋₄ alkyl, optionally substituted phenyl, C₁₋₁₂-aralkyloptionally substituted heteroaryl R¹² is H, —COAr¹, —CONH₂, —COOH,—COR¹⁶ or —CNHR⁶; the or each R¹³ is selected from OH, C₁₋₄ alkoxy, C₁₋₄alkyl, —NO₂, —NH₂, —NHR¹⁰, —NR¹⁰ ₂, —N⁺R¹⁰ ₃, —CN, Cl, Br, I, —NHCOR¹⁵,—COOH, —CONHR¹⁶, —NHCOOR¹⁶ and —COOR¹⁶; m is 0, 1 or 2; R¹⁴ is selectedfrom OH, C₁₋₄ alkoxy, C₁₋₄ alkyl, —NO₂, —NH₂, —CN, Cl, Br, I, —NHCOR¹⁵,—COOH, —CONHR¹⁶, —NHCOOR¹⁶, —COOR¹⁶ and H; R¹⁵ is selected from C₁₋₄alkyl, optionally substituted phenyl, optionally substituted heteroaryl,C₇₋₁₂ aralkyl Ar¹ and a ligand; R¹⁶ is selected from C₁₋₄ alkyl,optionally substituted phenyl, C₇₋₁₂-aralkyl, optionally substitutedheteroaryl and a ligand; and Ar¹⁰ is

in which x is 0, 1 or 2; Ar¹ is selected from the same groups as Arprovided that no more than one group R¹¹ or R¹³ in any one ring includesa group Ar¹.
 2. Use according to claim 1 in which the animal is a human.3. Use according to claim 1 or claim 2 in which the treatment is of atumour.
 4. Use according to any preceding claim in which Y is selectedfrom —OCOOR⁵, —OCONHR⁶, Cl, Br, and —OSOOR⁷, in which R⁵, R⁶ and R⁷ areindependently selected from C₁₋₄alkyl, optionally substituted phenyl,C₇₋₁₂-aralkyl and optionally substituted heteroaryl; preferably Cl. 5.Use according to any preceding claim in which Ar¹ is


6. Use according to any preceding claim in which R¹ is Ar.
 7. Useaccording to claim 6 in which Ar is a group


8. Use according to claim 7 in which n is at least one and one of thegroups R¹¹ of the Ar group is —NHCOAr¹.
 9. Use according to claim 8 inwhich Ar¹ is a group


10. Use according to claim 9 in which, in Ar¹, n is at least 2 and R¹¹is other than —NHCOAr¹, or n is
 0. 11. Use according to claim 6 in whichAr is a group


12. Use according to claim 11 in which R¹² is —COAr¹.
 13. Use accordingto claim 12 in which Ar¹ is a group


14. Use according to claim 13 in which, in Ar¹, R¹² is other than—COAr¹.
 15. Use according to any preceding claim in which R² is H. 16.Use according to any preceding claim in which R³ is H or COOMe.
 17. Useaccording to any preceding claim in which R⁴ is H.
 18. A compound of thegeneral formula I as defined in any of claims 1 and 4 to 17 for use inthe treatment an animal by therapy.
 19. A pharmaceutical compositioncomprising a compound of the general formula I as defined in any ofclaims 1 and 4 to 17 and a pharmaceutically acceptable excipient.
 20. Acompound of the general formula II or IIA or a salt thereof

in which R², R³ and R⁴ are as defined in claim 1 X¹ is H; Y¹ is aleaving group; R¹⁸ is H or an amine protecting group; R¹⁷ is R⁸, —OR⁸,NH₂ or Ar²; R⁸ is as defined in claim 1; Ar² is selected from

in which B¹ is N or CR⁴⁰, R⁴⁰ is selected from H, OH, C₁₋₄-alkoxy,C₁₋₄-alkyl, —NO₂, —NH₂, —CN, Cl, Br, I, —NHCOR²², —COOH, —CONHR²³,—NHCOOR²³ and —COOR²³; Z¹ is O, S, —CH═CH— or NR¹⁸; the or each R¹⁹ isselected from OH, C₁₋₄ alkoxy C₁₋₄ alkyl, NO₂, —NHR¹⁸, —NHR²³, —NR²³ ₂,—N⁺R²³ ₃, —CN, Cl, Br, I, —NHCOR²², —COOH, —CONHR²³ and —COOR²³; p is aninteger in the range 0 to 4; R²⁰ is H, COAr³, —CONH₂, —COOH —COR²³ or—COOR²³; the or each R²¹ is selected from OH, C₁₋₄ alkoxy C₁₋₄ alkyl,NO₂, —NHR¹⁸, —NHR²³, —NR²³ ₂, —N⁺R²³ ₃, —CN, Cl, Br, I, —NHCOR²², —COOH,—CONHR²³ and —COOR²³; q is 0, 1 or 2; R²² is selected from C₁₋₄ alkyl,optionally substituted phenyl, optionally substituted heteroalkyl, C₇₋₁₂aralkyl, Ar³ and ligands; R²³ is selected from C₁₋₄alkyl, optionallysubstituted phenyl, C₇₋₁₂-aralkyl and optionally substituted heteroaryland a ligand; and Ar³ is selected from the same groups as Ar² providedthat no more than one R¹⁹ or R²¹ in any one ring includes a group Ar³.21. A compound according to claim 20 in which the or each R¹⁸ is H. 22.A compound according to claim 20 or claim 21 in which Y¹ is selectedfrom —OCOOR⁵, —OCONHR⁶, Cl, Br, I, —OTos, or —OSOOR⁷ in which R⁵, R⁶ andR⁷ are as defined in claim 4, preferably Cl.
 23. A compound according toany of claims 20 to 22 in which Ar³ is


24. A compound according to any of claims 20 to 23 in which R¹⁷ is Ar².25. A compound according to claim 24 in which Ar² is a group


26. A compound according to claim 25 in which p is at least 1 and one ofthe groups R¹⁹ of the group R¹⁷ is a group —NHCOAr³.
 27. A compoundaccording to claim 26 in which Ar³ is


28. A compound according to claim 27 in which, in Ar³, p is at least 1and R¹⁹ is other than —NHCOAr³, or p is
 0. 29. A compound according toclaim 24 in which Ar² is a group


30. A compound according to claim 29 in which R²⁰ is COAr³.
 31. Acompound according to claim 30 in which Ar³ is


32. A compound according to claim 31 in which, in Ar³, R²⁰ is other than—COAr³.
 33. A compound according to any of claims 20 to 32 in which R²is H.
 34. A compound according to any of claims 20 to 33 in which R³ isH COOMe.
 35. A compound according to any of claims 20 to 34 in which R⁴is H.
 36. A compound according to claim 20 selectedfrom:—1-(chloromethyl)-6-benzoyl-3-((tert-butyloxy)carbonyl)-1,2-dihydro-3H-pyrrolo[3,2-e]indole;1-(chloromethyl)-6-benzoyl-3-((5-methoxy-1H-indol-2-yl)carbonyl)-1,2-dihydro-3H-pyrrolo[3,2-e]indole;1-(chloromethyl)-3-((5-methoxy-1H-indol-2-yl)carbonyl)-1,2-dihydro-3H-pyrrolo[3,2-e]indole;4-chloro-1-((tert-butyloxy)carbonyl)-1,2,3,4-tetrahydro-5,6-(9-benzoyl)pyrroloquinoline;4-chloro-1-(5-methoxy-1H-indol-2-ylcarbonyl)-1,2,3,4-tetrahydro-5,6-(9-benzoyl)pyrroloquinoline;4-chloro-1-(5-methoxy-1H-indol-2-ylcarbonyl)-1,2,3,4-tetrahydro-5,6)pyrroloquinoline;ethyl6-benzoyl-1-(chloromethyl)-3-((tert-butyloxy)carbonyl)-1,2-dihydro-3H-pyrrolo[3,2-e]indole-7-carboxylate;ethyl6-benzoyl-1-(chloromethyl)-3-((5-methoxy-1H-indol-2-yl)carbonyl)-1,2-dihydro-3H-pyrrolo[3,2-e]indole-7-carboxylate;ethyl1-(chloromethyl)-3-((5-methoxy-1H-indol-2-yl)carbonyl)-1,2-dihydro-3H-pyrrolo[3,2-e]indole-7-carboxylate;5,6-(9-benzoyl-8-(ethyloxy)carbonylpyrrolo)-1-((tert-butyloxy)carbonyl)-4-chloro-1,2,3,4-tetrahydroquinoline;5,6-(9-benzoyl-8-(ethyloxy)carbonylpyrrolo)-1-(5-methoxy-1H-indol-2-ylcarbonyl)-4-chloro-1,2,3,4-tetrahydroquinoline;and5,6(8-(ethyloxy)carbonylpyrrolo)-1-(5-methoxy-1H-indol-2-ylcarbonyl)-4-chloro-1,2,3,4-tetrahydroquinoline.37. A compound according to any of claims 20 to 36 for use in thetreatment of an animal by therapy.
 38. A pharmaceutical compositioncomprising a compound according to any claims 20 to 36 and apharmaceutically acceptable excipient.
 39. A compound of the generalformula III or IIIA

in which R² and R4 are as defined in claim 1; R³⁸ is selected from thesame groups as R³ defined in claim 1; X² is H; Y² is a leaving group ora hydroxyl or a protected hydroxyl group and R²⁴ and R²⁵ are each H oran amine protecting group.
 40. A compound according to claim 39 in whichR²⁴ and R²⁵ are different from one another.
 41. A compound according toclaim 40 in which R⁴ is BOC and R²⁵ is —COPh.
 42. A compound accordingto any of claims 39 to 41 in which Y² is selected from —OCOOR⁵,—OCONHR⁶, Cl, Br, and —OSOOR⁷, in which R⁵, R⁶ and R⁷ are independentlyselected from C₁₋₄ alkyl, optionally substituted phenyl, C₇₋₁₂-aralkyland optionally substituted heteroaryl; preferably Cl.
 43. A syntheticmethod in which a compound of the formula IV or IVA

in which R² and R⁴ are as defined in claim 1; R³⁷ is selected from H.C₁₋₄ alkyl, —OH, C₁₋₄ alkoxy, —CN, Cl, Br, I, —NO2, —NH₂, —NHCOR⁹,—COOH, —CONHR⁹, —NHCOOR⁹ and —COOR⁹; R⁹ is as defined in claim 1; X² andY² are as defined in claim 38; and R²⁶ is an amine protecting group; isreacted with a compound of the general formula V R²⁷COY³  V in which R²⁷is selected from C₁₋₄-alkyl, optionally substituted phenyl,C₇₋₁₂-aralkyl, optionally substituted heteroaryl and Ar⁴; Ar⁴ isselected from

in which B² is N or CR³²; Z² is O, S, —CH═CH— or NR³³; the or each R²⁸is selected from C₁₋₄-alkoxy, C₁₋₄-alkyl, NO₂, CN, Cl, Br, —NHR³³, —NR³⁶₂, —N⁺R³⁶ ₃—, —NHCOR³⁴, —COOH, —CONHR³⁵ and —COOR³⁵; r is an integer inthe range 0 to 4; R²⁹ is an amine protecting group; R³⁰ is an amineprotecting group, —CONH₂, —COOH, —COR³⁵ or —COAr⁵; the or each R³¹ isselected from C₁₋₄-alkoxy, C₁₋₄-alkyl, NO₂, CN, Cl, Br, —NHR³³, —NR³⁶ ₂,—N⁺R³⁶ ₃—, I, —NHCOR³⁴, —COOH, —CONHR³⁵ and —COOR³⁵; s is 0, 1 or 2; R³²is selected from H, C₁₋₄-alkoxy, C₁₋₄-alkyl, NO₂, CN, Cl, Br, I,NHCOR³⁴, —COOH, —CONHR³⁴, —NHCOOR³⁵ and COOR³⁴; the or each R³³ is anamine protecting group; R³⁴ is selected from Ar⁵, C₁₋₄-alkyl, optionallysubstituted phenyl, C₇₋₁₂-aralkyl, optionally substituted heteroaryl anda ligand; R³⁵ is selected from C₁₋₄-alkyl, optionally substitutedphenyl, C₇₋₁₂-aralkyl, optionally substituted heteroaryl and a ligand;each R³⁶ is selected from H, C₁₋₄ alkyl, optionally substituted phenyl,C₇₋₁₂ aralkyl and optionally substituted heteroaryl; Ar⁵ is selectedfrom the same groups as Ar⁴ and Y³ is a leaving group, provided that nomore than one R²⁸ or R³¹ is any one ring includes a group Ar⁵.
 44. Amethod according to claim 43 which is carried out in the presence of anamide coupling reagent.
 45. A method according to claim 42 or 43 inwhich the product is subsequently subjected to an amine deprotectionstep in which R²⁶, any or all groups R²⁹ (if any) and/or any or allgroups R³³ (if any) are replaced by H.
 46. A method according to any ofclaims 43 to 45 in which Y² is selected from —OCOOR⁵, —OCONHR⁶, Cl, Br,I, and —OSOOR⁷, in which R⁵, R⁶ and R⁷ are independently selected fromC₁₋₄ alkyl, optionally substituted phenyl, C₇₋₁₂-aralkyl and optionallysubstituted heteroaryl; preferably Cl.
 47. A method according to any ofclaims 43 to 46 in which Ar⁵ is


48. A method according to any of claims 43 to 47 in which R²⁷ is Ar⁴.49. A method according to claim 48 in which Ar⁴ is a group


50. A method according to claim 49 in which, in R²⁷, r is at least 1 andone of the groups R²⁸ is —NHCOAr⁵.
 51. A method according to claim 50 inwhich Ar⁵ is


52. A method according to claim 48 in which Ar⁴ is a group


53. A method according to claim 52 in which R³⁰ is —COAr⁵.
 54. A methodaccording to claim 53 in which, in R²⁷, Ar⁵ is a group


55. A method according to any of claims 43 to 54 in which the compoundof the formula IV or IVA is produced in a preliminary step including acyclisation step in which a compound of the general formula VI

in which R², R⁴, R²⁶, and X² are the same as in the compound of theformula IV; R³⁹ is the same as R³⁷ or is a precursor thereof; R⁴⁰ is anamine protecting group different from R²⁶; one of Z¹ and Z² is Y⁵ andthe other is H; Y⁵ hydrogen, or is a leaving group which is the same asor different to Y²; and Y⁴ is a radical leaving group is cyclised via anarylradical-alkene cyclisation step in the presence of a catalyst.
 56. Amethod according to claim 55 in which Z¹ is Y² and in which thecyclisation step is carried out in the presence of a free radical toform a dihydropyrrole ring.
 57. A method according to claim 56 in whichthe free radical is generated from azoisobutyronitrile or is a2,2,6,6-tetramethylpiperidinyloxy free radical.
 58. A method accordingto claim 56 or 57 in which the catalyst is tributyl tin hydride.
 59. Amethod according to claim 55 in which Z² is Y⁵, Y⁵ is a trialkyl tinradical, and the cyclisation step is carried out in the presence of apalladium complex to form a tetra hydroquinoline, which is oxidised toform an epoxide, the epoxide then being reduced to form an alcoholcompound and, if in the product of the formula IVA, Y² is other thanhydroxyl, the hydroxyl group of the alcohol compound is subsequentlyconverted into Y².
 60. A method according to any of claims 55 to 58 inwhich Y⁴ is a halogen atom, preferably Br or I.